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A novel androgen receptor-binding element modulates Cdc6 transcription in prostate cancer cells during cell-cycle progression.

ABSTRACT: The androgen receptor (AR) plays a pivotal role in the onset and progression of prostate cancer by promoting cellular proliferation. Recent studies suggest AR is a master regulator of G1-S progression and possibly a licensing factor for DNA replication yet the mechanisms remain poorly defined. Here we report that AR targets the human Cdc6 gene for transcriptional regulation. Cdc6 is an essential regulator of DNA replication in eukaryotic cells and its mRNA expression is inversely modulated by androgen or antiandrogen treatment in androgen-sensitive prostate cancer cells. AR binds at a distinct androgen-response element (ARE) in the Cdc6 promoter that is functionally required for androgen-dependent Cdc6 transcription. We found that peak AR occupancy at the novel ARE occurs during the G1/S phase concomitant with peak Cdc6 mRNA expression. We also identified several of the coactivators and corepressors involved in AR-dependent Cdc6 transcriptional regulation in vivo and further characterized ligand-induced alterations in histone acetylation and methylation at the Cdc6 promoter. Significantly, AR silencing in prostate cancer cells markedly decreases Cdc6 expression and androgen-dependent cellular proliferation. Collectively, our results suggest that Cdc6 is a key regulatory target for AR and provide new insights into the mechanisms of prostate cancer cell proliferation.

SUBMITTER: Jin F

PROVIDER: S-EPMC2724301 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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A novel androgen receptor-binding element modulates Cdc6 transcription in prostate cancer cells during cell-cycle progression.

Jin Feng F Fondell Joseph D JD

Nucleic acids research 20090611 14

The androgen receptor (AR) plays a pivotal role in the onset and progression of prostate cancer by promoting cellular proliferation. Recent studies suggest AR is a master regulator of G1-S progression and possibly a licensing factor for DNA replication yet the mechanisms remain poorly defined. Here we report that AR targets the human Cdc6 gene for transcriptional regulation. Cdc6 is an essential regulator of DNA replication in eukaryotic cells and its mRNA expression is inversely modulated by an ...[more]

PMID: 19520769

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Project description:BackgroundProstate cancer is a major cause of cancer morbidity and mortality in men worldwide. Androgen deprivation therapy (ADT) has proven effective in early-stage androgen-sensitive disease, but prostate cancer gradually develops into an androgen-resistant metastatic state in the vast majority of patients. According to our oncogene-induced model for cancer development, senescence is a major tumor progression barrier. However, whether senescence is implicated in the progression of early-stage androgen-sensitive to highly aggressive castration-resistant prostate cancer (CRPC) remains poorly addressed.MethodsAndrogen-dependent (LNCaP) and -independent (C4-2B and PC-3) cells were treated or not with enzalutamide, an Androgen Receptor (AR) inhibitor. RNA sequencing and pathway analyses were carried out in LNCaP cells to identify potential senescence regulators upon treatment. Assessment of the invasive potential of cells and senescence status following enzalutamide treatment and/or RNAi-mediated silencing of selected targets was performed in all cell lines, complemented by bioinformatics analyses on a wide range of in vitro and in vivo datasets. Key observations were validated in LNCaP and C4-2B mouse xenografts. Senescence induction was assessed by state-of-the-art GL13 staining by immunocytochemistry and confocal microscopy.ResultsWe demonstrate that enzalutamide treatment induces senescence in androgen-sensitive cells via reduction of the replication licensing factor CDC6. Mechanistically, we show that CDC6 downregulation is mediated through endogenous activation of the GATA2 transcription factor functioning as a CDC6 repressor. Intriguingly, GATA2 levels decrease in enzalutamide-resistant cells, leading to CDC6 stabilization accompanied by activation of Epithelial-To-Mesenchymal Transition (EMT) markers and absence of senescence. We show that CDC6 loss is sufficient to reverse oncogenic features and induce senescence regardless of treatment responsiveness, thereby identifying CDC6 as a critical determinant of prostate cancer progression.ConclusionsWe identify a key GATA2-CDC6 signaling axis which is reciprocally regulated in enzalutamide-sensitive and -resistant prostate cancer environments. Upon acquired resistance, GATA2 repression leads to CDC6 stabilization, with detrimental effects in disease progression through exacerbation of EMT and abrogation of senescence. However, bypassing the GATA2-CDC6 axis by direct inhibition of CDC6 reverses oncogenic features and establishes senescence, thereby offering a therapeutic window even after acquiring resistance to therapy.

Project description:The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.

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A novel androgen receptor-binding element modulates Cdc6 transcription in prostate cancer cells during cell-cycle progression.

Publications

A novel androgen receptor-binding element modulates Cdc6 transcription in prostate cancer cells during cell-cycle progression.

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